Microbiological studies of plaque-biofilms from toddlers reveal a direct association between early-childhood caries (ECC) and the presence of Candida albicans, along with high levels of Streptococcus mutans. Previous in vitro and in vivo studies suggest strongly that S. mutans interactions with C. albicans may influence the pathogenesis of ECC. Using a rodent model of the disease, enhanced levels of infection with elevated carriage of S. mutans and C. albicans were observed in plaque-biofilms from co-infected animals compared to those infected with either species alone. Importantly, the virulence of plaque-biofilm in co-infected animal was dramatically enhanced, leading to the development of rampant carious lesions on smooth-surface of teeth (similar to those found in ECC). Further in vitro studies have identified a cooperative and sucrose-dependent S. mutans-C. albicans interactions that is mediated by S. mutans exoenzymes termed glucosyltransferases (Gtfs). Of the three Gtfs, GtfB binds most avidly to C. albicans cell surface, retains enzymatic activity and produces exopolysaccharides (EPS) on C. albicans surfaces in the presence of sucrose in vitro. The surface-formed EPS enhances adhesive interactions between S. mutans and C. albicans, promotes the assembly of an EPS- rich extracellular matrix, and increases colonization of S. mutans and co-species biofilm formation onto saliva- coated apatitic surfaces. We hypothesize that S.mutans-C.albicans association mediated via GtfB modulates the development of hypercariogenic biofilms on teeth. It is possible that the assembly of EPS- rich matrix and elevated microbial accumulation enhance biofilm mechanical stability/cohesiveness and facilitate the creation of acidic microenvironments within biofilms, which can influence the onset and progression of caries in vivo. To test our hypothesis, we will (Aim 1) characterize the interaction of S. mutans derived-GtfB with C. albicans using genetic approaches (e.g. screening mutants of C. albicans) combined with spectroscopic/fluorescence and AFM methods (for GtfB binding and activity measurements) in vitro. GtfB binding and glucan synthesis by the enzyme on the fungal surface modulates S. mutans-C. albicans co- adherence and co-species biofilm formation. Therefore, we will identify C. albicans mutant strains that are defective in both the GtfB binding and enzymatic activity. In Aim 2, the effects of this fungal-bacterial interaction via GtfB on biofilm development and mechanical stability will be determined using a new engineering tool, while spatio-temporal development of acidic niches within biofilms will be assessed using time-lapsed pH mapping in vitro. Lastly, in Aim 3, we will examine the role of GtfB-mediated S. mutans-C. albicans interaction in the pathogenesis of the disease in vivo using GtfB-binding/activity defective C. albicans or gtfB-defective S. mutans (along with their parental strains) with our rodent model. A comprehensive program from laboratory studies to in vivo investigations is offered to provide critical insights into the mechanisms of this S. mutans-C albicans interaction and its implications in enhancing the virulence of dental caries disease.